JPH01250861A - Immunoassay of erythropoietin - Google Patents
Immunoassay of erythropoietinInfo
- Publication number
- JPH01250861A JPH01250861A JP63079868A JP7986888A JPH01250861A JP H01250861 A JPH01250861 A JP H01250861A JP 63079868 A JP63079868 A JP 63079868A JP 7986888 A JP7986888 A JP 7986888A JP H01250861 A JPH01250861 A JP H01250861A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- epo
- solid phase
- phase carrier
- conjugated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
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- 208000017733 acquired polycythemia vera Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
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- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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Abstract
Description
【発明の詳細な説明】
・崖】」≦１旧１褒Ｊ
本発明は、エリスロポエチン（以下ＥＰＯと略する）の
測定方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring erythropoietin (hereinafter abbreviated as EPO).
皿米二役街
ＥＰＯは、未熟な赤血球系前駆細胞に働き、分化増殖を
誘導する造血ホルモンの一つで、赤血球数を恒常的に保
つ上で非常に重要な役割を果たしている。すなわち、Ｅ
ＰＯは通常、組織の酵素需要に応じて主に腎臓で生産さ
れ、体内の赤血球量を調節する役割を担っている。低酸
素状態では、その産生が促進され、逆に高酸素状態では
低くおさえられている。Plate Rice Niyakugai EPO is one of the hematopoietic hormones that acts on immature erythroid progenitor cells and induces differentiation and proliferation, and plays a very important role in maintaining a constant number of red blood cells. That is, E
PO is normally produced primarily in the kidneys in response to tissue enzyme demands and plays a role in regulating the amount of red blood cells in the body. Its production is promoted under hypoxic conditions, and is suppressed under high oxygen conditions.
血中のＥＰＯ量を測定することは、Ｍｉ織における酸素
需要のバランスを知る上で非常に重要である。また、そ
の他臨床上、多数の有用な情報を得ることができる。例
えば、真性多血症及びり成性多血症は、臨床上類似し多
血症状を示すが、その原因が全く異なり、その治療方法
も異なっている。Measuring the amount of EPO in the blood is very important in understanding the balance of oxygen demand in Mi textiles. In addition, a large amount of other clinically useful information can be obtained. For example, polycythemia vera and polycythemia vera are clinically similar and exhibit polycythemia symptoms, but their causes are completely different and their treatment methods are also different.
すなわち、それらの症状における血中ＥＰＯ量は、前者
では非常に低値を示すのに対し、後者では、高値を示す
。したがって、血中ＥＰ○を測定することにより、両者
を容易に区別でき、適切な治療を行うことができる。ま
た、ＥＰＯを貧血患者に投与し、治療を行う場合におい
て、患者自身の血中ＢＰＯ量と外部から与えたＢＰＯ量
を追跡することは、適切な投与を行う上で非常に有用で
ある。That is, the amount of EPO in the blood in these symptoms shows a very low value in the former case, whereas it shows a high value in the latter case. Therefore, by measuring blood EP○, the two can be easily distinguished and appropriate treatment can be performed. Furthermore, when administering EPO to an anemic patient for treatment, it is very useful to track the amount of BPO in the patient's own blood and the amount of BPO given from an external source in order to perform appropriate administration.
この様に血中ＥＰＯの測定は臨床上非常に重要である。As described above, measurement of blood EPO is clinically very important.
ＢＰＯの測定には、マウスやラット等の小動物を用いる
バイオアッセイ法〔臨床検査技術全書第３巻血液検査、
小酒井望、阿部裕、林康之、古川俊之編、ｐ２２２．１
９７２ （医学書院）〕が一般的に知られていたが、該
方法は、測定操作が煩雑なうえに、測定感度が悪＜、臨
床応用には至らなかった。また、細胞培養技術の発達に
よって、骨髄細胞や胎児肝細胞の培養が可能となりｉｎ
ｖｉｔｒ。BPO is measured using a bioassay method using small animals such as mice and rats [Clinical Laboratory Techniques Volume 3 Blood Test,
Edited by Nozomi Kozakai, Yutaka Abe, Yasuyuki Hayashi, and Toshiyuki Furukawa, p222.1
972 (Igaku Shoin)] was generally known, but this method required complicated measurement operations and had poor measurement sensitivity, so it was not clinically applicable. Furthermore, advances in cell culture technology have made it possible to culture bone marrow cells and fetal liver cells in vitro.
vitr.
バイオアッセイ法（Ｅ、Ｇｏｌｄｗａｓｓｅｒ ａｎｄ
Ｇｒｏｓｓ、 ｒメソオズオブエンザイモロジーＪ
（Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏ＋、）Ｘ Ｘ Ｘ Ｖ
ＩＩ 、ｐ１０９ （１９７５）） 、 （Ｎ、
Ｃ，Ｂｒａｎｄａｎ、Ｐ、Ｍ。Bioassay method (E, Goldwasser and
Gross, rMethods of Enzymology J
(Methods Enzymo+,)
II, p109 (1975)), (N,
C,Brandan,P,M.
Ｃｏｔｅｓ ａｎｄ Ｊ、Ｅｓｐａｄａ、 ｒブリテ
ィッシュジャーナルオブヘマトロジーＪ （Ｂｒ、Ｊ、
Ｈａｅｍａｔｏｌ ）４Ｌ４６１ （１９８１） ）が
確立し、測定感度の改善が行われたが、試料中の夾雑物
の影響を受けやすく、煩雑な操作を要するため、やはり
臨床応用には至らなかった。Cotes and J, Espada, r British Journal of Hematology J (Br, J,
Haematol ) 4L461 (1981)) was established, and the measurement sensitivity was improved, but it was still not clinically applicable because it was easily affected by contaminants in the sample and required complicated operations.
また、すでに、ＢＰＯと特異的に反応する抗体（以下特
異抗体と呼ぶ）を用いた免疫学的な測定法であるラジオ
イムノアッセイ法（ＲＩＡ法）を確立し［１１，Ｍｉｚ
ｏｇｕｃｈｉ ｅｔ ａｌ、 ｒアクタヘマトールジャ
バンＪ （Ａｃｔａ Ｈａｅｎ＋ａｔｏｌ Ｊｐｎ、
５０．１５ （１９８７））正常血中ＥＰＯの測定を可
能とした。しかし、該方法では、放射性同位元素（＋２
５１）を用いなければならず、そのうえ特殊施設を要し
、廃棄物処理上の問題点を有していた。さらには、近年
、酵素免疫測定法が著しく進歩し、血中ＥＰＯを測定し
ようとする試みがなされているが、正常人血中ＢＰＯ量
が１（１−２０ｍ Ｕ／ｍ ｊ！ （０，１〜０．２ｎ
ｇ／ｍ Ｉり と極めて微量であるため、公知の酵素免
疫測定法で測定するには、検出が困難であると考えられ
ていた。Furthermore, we have already established the radioimmunoassay method (RIA method), which is an immunological measurement method using antibodies that specifically react with BPO (hereinafter referred to as specific antibodies) [11, Miz
Oguchi et al, Acta Haen+Atol Jpn,
50.15 (1987)) enabled the measurement of normal blood EPO. However, in this method, radioactive isotope (+2
51), special facilities were required, and there were problems in waste disposal. Furthermore, in recent years, enzyme immunoassay has made remarkable progress, and attempts have been made to measure blood EPO, but it has been found that the amount of BPO in the blood of normal people is 1 (1-20 m U/m j! (0,1 ~0.2n
Since the amount is extremely small (g/ml), it was thought that it would be difficult to detect using the known enzyme immunoassay method.
このため、本発明者らは、さきに、ＥＰＯの特異抗体を
第１抗体として不溶性支持体に結合させた抗体結合不溶
性支持体にＥＰＯを含む被検液及びＥＰＯの特異抗体で
且つ前記第１抗体とは異なる動物種に免役して得られた
第２抗体を反応させた後、さらに、前記第２抗体を作製
した動物種の免疫グロブリンと特異的に反応する第３抗
体に酵素標識を作った標識抗体を反応させ、前記抗体結
合不溶性支持体上に結合した前記標識抗体の量を測定す
るか、もしくは結合しなかった標識抗体の量を測定する
ことにより前記被検液中のＢＰＯ量を測定する免疫学的
測定法を完成し、既に特許出願した（特願昭６２Ｊｌ夛
）。しかし、この技術は、非常に煩雑な操作を有すると
いう欠点があった。さらには、高感度測定が可能な測定
法として、グルコース−６−リン酸脱水素酵素を用いた
標識抗体を作製し、グルコース−６−リン酸脱水素酵素
の活性測定を生物発光法で行う、サンドイッチＥＩＡ法
（エンザイムイムノアツセイ法）を完成し、既に特許出
願した（欅願昭６２−２３５５６６号）。For this reason, the present inventors first prepared a test solution containing EPO and a test solution containing EPO on an antibody-bonded insoluble support in which an EPO-specific antibody was bound to an insoluble support as a first antibody, and the first antibody was bound to an insoluble support. After reacting with a second antibody obtained by immunization with an animal species different from the antibody, an enzyme label is created in a third antibody that specifically reacts with the immunoglobulin of the animal species in which the second antibody was produced. The amount of BPO in the test liquid is determined by reacting the labeled antibody and measuring the amount of the labeled antibody bound to the antibody-bound insoluble support, or by measuring the amount of labeled antibody that is not bound. He completed an immunoassay method and has already applied for a patent (patent application published in 1982). However, this technique has the drawback of requiring very complicated operations. Furthermore, as a measurement method that allows for highly sensitive measurements, a labeled antibody using glucose-6-phosphate dehydrogenase is prepared, and the activity of glucose-6-phosphate dehydrogenase is measured using a bioluminescence method. We have completed the sandwich EIA method (enzyme immunoassay method) and have already applied for a patent (Keyaki patent application No. 62-235566).
しかし、該方法においても、生物発光測定というやや特
殊測定装置を要するという欠点を有しており、さらには
、上記ＥＩＡ法においてはポリクローナル抗体を使用す
るために作製したポリクローナル抗体のロフトが変わる
ごとに感度が変わるという不利な点があった。However, this method also has the disadvantage of requiring a somewhat special measuring device called bioluminescence measurement.Furthermore, in the EIA method described above, each time the loft of the polyclonal antibody prepared to use the polyclonal antibody changes, The disadvantage was that the sensitivity changed.
発註が解決しようとする課題
本発明者らは、畝上の状況に鑑み、使用するモノクロー
ナル抗体としてエピトープの異なる２種の抗体を選択し
することにより、煩雑な操作を必要とせず、或は、特殊
な装置を必要とせず、通常の臨床検査に用いるイムノリ
ーダーを用いて短時間に実施でき、且つ検査感度の高い
ＥＰＯのサンドイッチＥＩＡ法による測定方法を堤供す
ることを課題とする。Problems to be Solved by the Invention In view of the situation, the present inventors selected two types of antibodies with different epitopes as monoclonal antibodies to be used, thereby eliminating the need for complicated operations or It is an object of the present invention to provide a method for measuring EPO using a sandwich EIA method that does not require special equipment, can be carried out in a short time using an immunoreader used in ordinary clinical tests, and has high test sensitivity.
本発明の特徴は、ＥＰＯに対するエピトープの異なる２
種のモノクローナル抗体を選択し、ＥＰＯと特異的に反
応する第１抗体を固相担体く例えば、ポリ塩化ビニール
プラスチック製９６穴フレキシブルアセイブレート、フ
ァルコン社製、Ｆａｌｃｏｎ３９１２）に結合させた抗
体結合固相担体に、ＥＰＯを含む被検液を加えて反応さ
せ、抗体結合固相担体にＥＰＯを結合させた後、前記第
１抗体とは異１〆
なる抗体決定部位を有する第２抗体に標識物質（例えば
、アルカリフォスファターゼ、Ｓｉｇｍａ Ａｌ−ｋａ
ｌｉｎｅ ｐｈｏｓｐｈａｔａｓｅ Ｔｙｐｅ Ｖ Ｕ
−Ｓウシ小腸由来Ｐ−５５２１）を結合させた標識抗体
を反応させ、前記抗体結合固相担体上に結合した前記標
識抗体の量を測定することにより前記被検液中のＥＰＯ
量を測定することから構成されるＥＰＯの免疫学的測定
法にある。The present invention is characterized by two different epitopes for EPO.
A monoclonal antibody of a species is selected, and a first antibody that specifically reacts with EPO is bonded to a solid phase carrier (for example, a 96-hole flexible asylate made of polyvinyl chloride plastic, Falcon 3912). A test solution containing EPO is added to the phase carrier and reacted to bind EPO to the antibody-bound solid phase carrier, and then a labeling substance is added to the second antibody, which has an antibody determining site different from that of the first antibody. (e.g. alkaline phosphatase, Sigma Al-ka
line phosphotase Type V U
-S bovine small intestine-derived P-5521) is reacted with a labeled antibody, and the amount of the labeled antibody bound to the antibody-bound solid phase carrier is measured.
An immunoassay for EPO consists of measuring the amount of EPO.
立朋
本発明は、上述の如く構成されるが、要は、ＥＰＯと特
異的に反応する第１抗体を結合した抗体結合固相担体に
抗原であるＥＰＯを介して、標識物質を結合させた第２
抗体を結合させ、結合した標識物質を測定することによ
り非常に簡便に血中等のｉｔにしか存在しないＥＰＯを
感度よく測定することを可能としたものである。The present invention is constructed as described above, but the point is that a labeling substance is bound to an antibody-bound solid phase carrier to which a first antibody that specifically reacts with EPO is bound via EPO, which is an antigen. Second
By binding an antibody and measuring the bound labeled substance, it is possible to very easily and sensitively measure EPO, which exists only in IT, such as blood.
本発明に使用する特異性の高い抗体を得るには、ＥＰＯ
をできるだけ純化しておくことが好ましい。To obtain highly specific antibodies used in the present invention, EPO
It is preferable to purify as much as possible.
例えば、アフィニティクロマトグラフゲル濾過等の公知
の精製手段を組合わせて実施可能である。For example, it can be carried out in combination with known purification means such as affinity chromatography gel filtration.
この精製ＥＰＯをＢａ１ｂ／ｃマウスに免疫して牌細胞
を得、骨髄腫細胞とポリエチレングリコールにより融合
させ、ＨＡＴ選択培地による選別と限界希釈法によるク
ローニングをくり返すことにより得られる。Balb/c mice are immunized with this purified EPO to obtain tile cells, which are fused with myeloma cells using polyethylene glycol, followed by repeated selection with HAT selection medium and cloning by limiting dilution.
次に、この様にして得られた第１抗体を固相担体に結合
させる。固相担体としては、マイクロプレート、ポリス
チレンチューブ、ポリスチレン球、シリコーン片などが
あげられるが、特にポリ塩化ビニールプラスチック製９
６六マイクロプレートが好ましい。これらの固相担体に
結合させる方法は、公知の化学的方法もしくは物理的方
法のいずれの方法でもよい。例えば、物理的方法として
は、抗体を適当な緩衝液に熔解し、前記固相担体を接触
させて、０℃〜室温にて、数時間から一夜、好ましくは
、４℃、−夜或は室温２時間放置した後、Ｔｗｅｅｎを
含むＰＢＳ （リン酸緩衝生理的食塩水；ｐｈｏｓｐｈ
ａｔｅ Ｂｕｆｆｅｒｄ ５ａｌｉｎｅ）にて洗浄を行
い未吸着抗体を除去した後、牛血清アルブミンを含むＰ
ＢＳとＯ℃〜室温にて、数時間から一夜、好ましくは、
４℃−夜、或は室温２時間接触させることにより、未反
応の固相担体活性基に牛血清アルブミンを吸着させた後
、前述のＴｗｅｅｎを含むＰＢＳ溶液にて再び洗浄する
。この様にして、抗体結合固相担体を作製する。Next, the first antibody thus obtained is bound to a solid phase carrier. Examples of the solid support include microplates, polystyrene tubes, polystyrene spheres, silicone pieces, and in particular, polyvinyl chloride plastic 9
66 microplates are preferred. The method of binding to these solid phase carriers may be any known chemical method or physical method. For example, as a physical method, the antibody is dissolved in a suitable buffer solution and brought into contact with the solid phase carrier, and the temperature is kept at 0°C to room temperature for several hours to overnight, preferably at 4°C, overnight or at room temperature. After leaving for 2 hours, PBS (phosphate buffered saline; phospho
ate Buffer 5aline) to remove unadsorbed antibodies, P containing bovine serum albumin was added.
BS and at 0°C to room temperature for several hours to overnight, preferably.
Bovine serum albumin is adsorbed onto the unreacted active groups of the solid phase carrier by contacting at 4° C. overnight or at room temperature for 2 hours, and then washed again with the above-mentioned PBS solution containing Tween. In this way, an antibody-bound solid phase carrier is produced.
次に、第２抗体に結合させた標識抗体（以下単に標識抗
体と呼ぶ）を作製する。第２抗体は第１抗体とは抗原決
定部位（抗原の認識結合部位）と異なる抗原決定部を存
する抗体を用いて行う。第２抗体と標識物質の結合は公
知の方法で実施できる。例えば、グルタルアルデヒドを
用いて行い得る。すなわち、第２抗体溶液と標識物質溶
液を混合し、グルタルアルデヒドを加え、室温で１〜２
時間反応させた後、０〜４℃で一晩透析し、未反応のグ
ルタルアルデヒドを除去してのち、トリス緩衝液にて、
さらに、０〜４℃で一晩透析し、未反応のグルタルアル
デヒドを不活性化する。尚、透析は好ましくは、透析外
液を１〜３回途中で取り換える。Next, a labeled antibody (hereinafter simply referred to as labeled antibody) bound to the second antibody is produced. The second antibody is an antibody that has an antigen-determining site (antigen recognition and binding site) different from that of the first antibody. Binding of the second antibody and the labeling substance can be carried out by a known method. For example, this can be done using glutaraldehyde. That is, the second antibody solution and the labeling substance solution were mixed, glutaraldehyde was added, and the mixture was incubated at room temperature for 1 to 2 hours.
After reacting for an hour, dialysis was performed overnight at 0 to 4°C to remove unreacted glutaraldehyde, and then with Tris buffer.
Further, unreacted glutaraldehyde is inactivated by dialysis overnight at 0 to 4°C. Note that during dialysis, the external dialysis solution is preferably replaced 1 to 3 times.
以上のようにして調製した標識第２抗体に安定剤である
牛血清アルブミン及び窒化ナトリウムを加え、冷暗所に
保存すれば、少くとも６ケ月は安定である。ここで標識
物質としては、酵素、螢光物質、金属などがあげられる
が特に酵素が好ましい。さらに酵素としては、アルカリ
フォスファターゼ、西洋ワサビペルオキシダーゼ、β−
ガラクトシダーゼ、グルコースオキシダーゼ、グルコー
ス−６−リン酸デヒドロゲナーゼ、グルコースデヒドロ
ゲナーゼなどが好適であるが、特にアルカリフォスファ
ターゼが有利である。If stabilizers such as bovine serum albumin and sodium nitride are added to the labeled second antibody prepared as described above, and the antibody is stored in a cool, dark place, it will be stable for at least 6 months. Examples of the labeling substance include enzymes, fluorescent substances, metals, etc., and enzymes are particularly preferred. Furthermore, the enzymes include alkaline phosphatase, horseradish peroxidase, β-
Galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase, etc. are suitable, and alkaline phosphatase is particularly advantageous.
以上の様にして得られた抗体結合固相担体及び標識抗体
を用いてＥＰＯを測定するには、次の様にして行う。ま
ず抗体結合固相担体にＥＰＯを含む被検液を加え０〜５
０℃にて１時間〜１夜、好ましくは、１５〜３５℃にて
２〜４時間反応させる。ここでＥＰＯを含む被検液とは
、血清、血漿、尿、ＥＰＯ生産培養液などを示す。この
様にして反応させた抗体結合固相担体上には、被検液中
に含まれるＥＰＯｉに比例してＥＰＯが結合しているの
で、抗体結合固相担体を洗浄後、更に標識抗体を０〜５
０℃にて１時間〜１夜、好ましくは、１５〜３５℃にて
２〜４時間反応させると前記抗体結合固相担体上にはＥ
ＰＯの結合量に比例して標識抗体が結合することになる
。したがって、この標識抗体の標識物質の量を測定し、
予め作成した検量線より被検液中のＥＰＯを測定するこ
とができる。ここで標識物質として酵素を用いた場合は
、その酵素活性を測定すればよい。Measurement of EPO using the antibody-bound solid phase carrier and labeled antibody obtained as described above is carried out as follows. First, add a test solution containing EPO to the antibody-bound solid phase carrier and
The reaction is carried out at 0°C for 1 hour to overnight, preferably at 15 to 35°C for 2 to 4 hours. Here, the test liquid containing EPO refers to serum, plasma, urine, EPO production culture solution, and the like. EPO is bound on the antibody-bound solid phase carrier reacted in this way in proportion to the EPOi contained in the test solution, so after washing the antibody-bound solid phase carrier, the labeled antibody is further removed. ~5
When reacted at 0°C for 1 hour to overnight, preferably at 15 to 35°C for 2 to 4 hours, E.
The labeled antibody will bind in proportion to the amount of PO bound. Therefore, the amount of labeled substance in this labeled antibody is measured,
EPO in the test liquid can be measured using a calibration curve prepared in advance. When an enzyme is used as a labeling substance, the enzyme activity may be measured.
また、上記のＥＰＯの測定操作をより筒便に行うには、
標識抗体に使用する第２抗体にモノクローナル抗体を使
用しているため、抗体結合固相担体とＥＰＯ含有の被検
液及び標識抗体を同時に混合し、０〜５０℃にて１時間
〜１夜、好ましくは、１５〜３５℃にて２〜４時間反応
後、前記と同様にして抗体結合固相担体に結合した標識
物質の量を測定し、予め作成した検量線より被検液中の
ＥＰＯをより簡便に測定することができる。In addition, in order to perform the above EPO measurement operation more conveniently,
Since a monoclonal antibody is used as the second antibody used for the labeled antibody, the antibody-bound solid phase carrier, the EPO-containing test solution, and the labeled antibody are simultaneously mixed and incubated at 0 to 50°C for 1 hour to overnight. Preferably, after reacting at 15 to 35°C for 2 to 4 hours, the amount of labeling substance bound to the antibody-bound solid phase carrier is measured in the same manner as above, and the EPO in the test solution is determined using a calibration curve prepared in advance. It can be measured more easily.
以下、実施例により本発明を説明するが、本発明は、こ
れらの実施例により限定されるものではない。EXAMPLES The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples.
実施例１
■抗原ＥＰＯの調製：
特開昭６０−４１６１４号公報に記載の方法に従って、
貧血愚者尿より分離したＥＰＯ（貧血患者尿濃縮物をＳ
ＤＳ処理後、抗体吸着処理及びゲル濾過により取得した
ＥＰＯ標品）のＰＢＳ溶解物を水冷し、−２０°Ｃに冷
却した９９．５％エタノールの９倍量を添加してＥＰＯ
を沈澱させた。この沈澱を一２０℃の９０％エタノール
溶液で洗浄した後、減圧下に乾燥し、０．０１ｍＭ Ｃ
ａＣＩｚを含む１０ｍＭ ＮａＰｉバッファー（ｐＨ６
，８）に熔解し、予め同じバッファーで平衡化したハイ
ドロキシアパタイトカラムに通し、非吸着画分にＥＰＯ
を回収した。得られたＥＰＯの純度は９９％であった。Example 1 ■Preparation of antigen EPO: According to the method described in JP-A-60-41614,
EPO isolated from anemic patient urine (anemic patient urine concentrate is
After DS treatment, the PBS solution of the EPO preparation (obtained by antibody adsorption treatment and gel filtration) was cooled with water, and 9 times the volume of 99.5% ethanol cooled to -20°C was added to prepare EPO.
was precipitated. This precipitate was washed with a 90% ethanol solution at -20°C, dried under reduced pressure, and diluted with 0.01mM C
10mM NaPi buffer (pH 6) containing aCIz
, 8), passed through a hydroxyapatite column equilibrated with the same buffer, and added EPO to the non-adsorbed fraction.
was recovered. The purity of the obtained EPO was 99%.
■上記ＥＰＯによる 験Φ の ：
前記の方法にて調製した純化ＥＰ○を抗原として使用し
、実験動物としてマウス（Ｂａｌｂ／ｃマウス）を用い
、このマウスに対し、次のとおり２週間間隔で３回免疫
を行った。■Experiment Φ with the above EPO: Purified EP○ prepared by the above method was used as an antigen, and a mouse (Balb/c mouse) was used as an experimental animal. Immunization was performed.
第１回免疫：
ＰＢＳ中に純化ＥＰＯクンバク質を１ｍｇ／ｍ ｉｔで
溶解し、これに等量のフロイント完全アジュバントを混
合して得たエマルジョン０．２ｍ７！をマウスに対し腹
腔内注射で投与した。1st immunization: 0.2 ml of emulsion obtained by dissolving purified EPO Kumbaku substance in PBS at 1 mg/m it and mixing it with an equal amount of Freund's complete adjuvant. was administered to mice by intraperitoneal injection.
第２回免疫：
２週間後に同上のエマルジョン液１００μａをマウスに
対し腹腔内注射で投与した。Second immunization: Two weeks later, 100 μa of the above emulsion was administered to the mice by intraperitoneal injection.
第３回免疫：
ＰＢＳ中に純化ＥＰＯを０．５ｍｇ／ｍ １！で溶解し
、１００μｌをマウスに対し、２週間後に腹腔的投与し
た。3rd immunization: 0.5mg/m of purified EPO in PBS 1! 100 μl was intraperitoneally administered to mice 2 weeks later.
■抗ＥＰＯ抗体産生ハイプリドーマの調製：ｉ）細胞融
合の作製
前記免疫処理終了から３日後に免疫マウスの肺細胞を無
菌的に摘出し、合成培養液（ＲＰＭ１１６４０液）と１
５％生胎児血清（ＦＣＳ）との混合液で洗浄後、該混合
液中で肺臓細胞をハサミで細断して単細胞化を行い、該
混合液で２回洗浄した後、単細胞化した細胞をＲＰ Ｍ
ｒ １６４０液に分散した。細胞数は８ Ｘ １０’
個であった。別にマウスのミエローマ細胞（Ｐ３／ＮＳ
Ｉ／１−Ａｇ４−１）を前記ＲＰＭＩ及びＦＣＳの混合
溶液中で培養し、増殖した細胞をＲＰＭ Ｉ １６４０
液で洗浄した。細胞数は４ Ｘ １０”個であった。■ Preparation of anti-EPO antibody-producing hybridomas: i) Preparation of cell fusion Three days after the completion of the immunization, lung cells from the immunized mice were aseptically removed and mixed with synthetic culture medium (RPM11640 solution).
After washing with a mixture of 5% live fetal serum (FCS), the lung cells were shredded with scissors in the mixture to make them into single cells, and after washing twice with the mixture, the cells were made into single cells. RPM
Dispersed in r 1640 liquid. Number of cells is 8 x 10'
It was. Separately, mouse myeloma cells (P3/NS
I/1-Ag4-1) was cultured in the mixed solution of RPMI and FCS, and the proliferated cells were cultured in RPM I 1640.
Washed with liquid. Cell number was 4 x 10''.
次に前記で調製した免疫マウス牌細胞とマウスミエロー
マ細胞とをＲＰ Ｍ １１６４０液に分散し、混合した
後、遠心し、上滑を除去した。混合細胞をポリエチレン
グリコール１５００の５０％溶液中で細胞融合させた後
、融合細胞をＨＴ培養液（ヒボキサンチン、チミジン及
び１５％牛脂児血清を含むＲＰ Ｍ Ｉ １６４０液）
に混合し、混合液を８枚の９６穴マイクロタイタープレ
ートにまいて２日目以降、ＨＡＴ培養液（ヒボキサンチ
ン、アミノプテリン、チミジン及び１５％牛脂児血清を
含むＲＰＭ１１６４０液）を添加して、各人で２週間培
養してＨＡＴ選択を行った。増殖したハイブリドーマ細
胞を３２６穴において確認した。Next, the immunized mouse tile cells and mouse myeloma cells prepared above were dispersed in RPM 11640 solution, mixed, and then centrifuged to remove the supernatant. After fusion of the mixed cells in a 50% solution of polyethylene glycol 1500, the fused cells were placed in HT culture medium (RP M I 1640 solution containing hyboxanthin, thymidine, and 15% tallow serum).
The mixture was spread on eight 96-well microtiter plates, and from the second day onwards, HAT culture solution (RPM11640 solution containing hypoxanthin, aminopterin, thymidine, and 15% tallow serum) was added to each plate. HAT selection was performed by culturing in humans for 2 weeks. Proliferated hybridoma cells were confirmed in 326 wells.
ＩＩ）上記ハイブリドーマ細胞のスクリーニングによる
選出
前記で得た３２６種類のハイブリドーマより特定の細胞
、すなわち、ＥＰＯと特異的に結合し得る抗体を産生ず
るハイブリドーマを選び出すために、”’Ｉ−ＥＰＯ（
ヒト尿由来純化ＥＰＯをｌ０Ｄ０−ＧＥＮ法にて１２５
１で標識したも）９８μｃｉ／μｇＥＰ○）を用いて＋
２５ｒ ＥＰＯと特異的に結合する抗体が、ハイブリ
ドーマ培養液中に存在することを確認して選出した。II) Selection by screening the above hybridoma cells In order to select specific cells from the 326 types of hybridomas obtained above, that is, hybridomas that produce antibodies that can specifically bind to EPO, "'I-EPO (
Purified EPO derived from human urine was purified using the 10D0-GEN method.
1) using 98 μci/μg EP○)
An antibody that specifically binds to 25r EPO was selected after confirming its presence in the hybridoma culture medium.
その結果、目的に適合したものとして２２種類の細胞が
選出され、そのうち１２Ｊ−ＥＰＯとの結合値の高かっ
たもの８種についてハイブリドーマの継代培養を続け、
限界希釈法によりモノクローン化を行い、安定的に抗体
産出を行う４種のハイブリドーマ細胞（門Ｃ−Ｒ−２、
ＭＣ−Ｒ−４、肛−Ｒ−６、ＭＣ−１？−１２）、を得
た。As a result, 22 types of cells were selected as suitable for the purpose, and among them, 8 types with high binding values with 12J-EPO were continued to be subcultured into hybridomas.
Four types of hybridoma cells (C-R-2, C-R-2,
MC-R-4, anal-R-6, MC-1? -12) was obtained.
■モノクローナル抗ＥＰＯ抗体の生産：前記のハイブリ
ドーマ細胞ＭＣ−１？−２、ＭＣ−Ｒ−４、ＭＣ−Ｒ−
６、ＭＣ−Ｒ−１２を用いて、抗体産生を行った。■Production of monoclonal anti-EPO antibody: the above hybridoma cell MC-1? -2, MC-R-4, MC-R-
6. Antibody production was performed using MC-R-12.
すなわち、常法通りに、マウス腹腔内に、各細胞を移植
し、マウス腹腔内で抗体を生産させ、５０％硫安分画に
付した後、ＤＥ５２（ワットマン社製、ＤＥＡＥ−セル
ロース）充填カラムを通し、０．１Ｍ〜０．２Ｍ Ｎａ
Ｃｌ画分として精製免疫グロブリン（ＩｇＧ）を得た。That is, each cell was transplanted into the peritoneal cavity of a mouse in the usual manner, antibodies were produced in the mouse peritoneal cavity, and after being subjected to 50% ammonium sulfate fractionation, a column packed with DE52 (manufactured by Whatman, DEAE-cellulose) was inserted. Through, 0.1M~0.2M Na
Purified immunoglobulin (IgG) was obtained as a Cl fraction.
各細胞当りマウス１０匹を用い、それぞれ３００ｍｇ、
２８０ｍｇ、２５０ｍｇ、２６０ｍｇの精製モノクロ
ーナル抗体を得た。Using 10 mice per each cell, 300 mg each,
280 mg, 250 mg, and 260 mg of purified monoclonal antibodies were obtained.
■抗体結合支持体の作成：
ポリ塩化ビニ〜ルプラスチック製９６穴フレキシブルア
ッセイプレート （ファルコン社製、Ｆａｒｃｏｎ３９
１２）をエタノールで洗浄し、乾燥した後に抗ＥＰＯモ
ノクローナル抗体ＭＣ−Ｒ−６を１０ ｐ ｇ’／ｌｎ
ｊ！になるように０．０５Ｍ炭酸ナトリウム緩衝液（
ｐｔ＋ ９．６）に溶解した抗体液をアッセイプレート
の各ウェルに１００μｌずつ分注し、４℃１晩（或は室
温２時間）静置後、抗体溶液を除去し、Ｐ Ｂ Ｓ −
Ｔｗｅｅｎ溶液（０，０５％Ｔｗｅｅｎ２０．０．０２
％ＮａＮ１含有ＰＢＳ溶液）でウェル内を３回洗浄した
後に、各ウェルに１％ＢＳＡ−ＰＢＳ溶液（１％ＢＳＡ
含有ＰＢＳ溶液）を２００μβずつ分注し、室温で２時
間静置後、ウェル内をＰ Ｂ Ｓ−Ｔｗｅｅｎ溶液で３
回洗浄し、抗体コートウェル（抗体結合支持体）とした
。■Preparation of antibody-binding support: 96-well flexible assay plate made of polyvinyl chloride plastic (Falcon 39, manufactured by Falcon)
12) was washed with ethanol, dried, and then treated with anti-EPO monoclonal antibody MC-R-6 at 10 p g'/ln.
j! Add 0.05M sodium carbonate buffer (
Pipette 100 μl of the antibody solution dissolved in Pt+ 9.6) into each well of the assay plate, leave it at 4°C overnight (or 2 hours at room temperature), remove the antibody solution, and add PBS-
Tween solution (0.05% Tween20.0.02
After washing the inside of the well three times with 1% BSA-PBS solution (1% BSA-containing PBS solution) in each well
After dispensing 200 μβ of each PBS solution (PBS solution containing
It was washed twice and used as an antibody-coated well (antibody-binding support).
■酵素標識抗体の作成：
抗体として抗ＥＰＯモノクローナル抗体ＭＣ−Ｒ−２を
、酵素としてシグマ社製ウシ小腸由来アルカリフォスフ
ァターゼ（Ｓｉｇｍａ Ａｌｋａｌｉｎｅ Ｐｈｏｓｐ
ｈａｔａｓｅＴｙｐｅ■−８）を用いた。■Preparation of enzyme-labeled antibody: Anti-EPO monoclonal antibody MC-R-2 was used as the antibody, and alkaline phosphatase derived from bovine small intestine (Sigma Alkaline Phosp) was used as the enzyme.
hataseType ■-8) was used.
１．４ｍｇのＭＣ−Ｒ−２と５０００単位のアルカリフ
ォスファターゼを０．０５Ｍ Ｐ Ｂ Ｓ （０，１５
？Ｌ ＮａＣｌを含む５０ｍＭリン酸ナトリウム緩衝ｉ
）ｔｍｌに溶解し、４“Ｃで一晩透析する。尚、透析外
液は０．０５Ｍ ＰＢＳ ＩＮで、適時２回の外液交換
を行った。次に２５％グルタルアルデヒド溶液を０．２
％（ν／ν）になる様に加え、室温で１〜２時間反応さ
せた後に、４℃で一晩透析を行った。尚、透析外液は０
．０５Ｍ ＰＢＳ ＩＮで、適時２回の外液交換を行っ
た。さらに透析外液を５０１１１Ｍトリス緩衝液（ｐＨ
８，０）、１ｍＭ ＭｇＣＩｚに交換し、４℃で一晩透
析する（外液は５００ｍβで適時２回交換）。透析終了
後、ＢＳＡ １％（ｗ／ｖ）、ＮａＮ３０．０２％（ｗ
／ｖ） となる様に５％ＢＳＡ、０．１％ＮａＮ、、
１ｍＭ ＭｇＣＩｇ含有５０ｍＭ トリス緩衝液を１７
４容量加え、酵素標識抗体溶液とした。尚、本溶液は、
４℃暗所で保存した。1.4 mg of MC-R-2 and 5000 units of alkaline phosphatase were mixed in 0.05M PBS (0,15
? 50mM sodium phosphate buffer containing L NaCl
) tml and dialyzed overnight at 4"C. The external dialysis solution was 0.05M PBS IN, and the external solution was exchanged twice at appropriate times. Next, 25% glutaraldehyde solution was diluted with 0.2% glutaraldehyde solution.
% (v/v), and after reacting at room temperature for 1 to 2 hours, dialysis was performed at 4°C overnight. In addition, the external dialysis fluid is 0.
．． At 05M PBS IN, external fluid was exchanged twice at appropriate times. Furthermore, the external dialysis solution was added to 50111M Tris buffer (pH
8,0), exchanged with 1mM MgCIz and dialyzed overnight at 4°C (external solution was exchanged twice at 500mβ). After dialysis, BSA 1% (w/v), NaN 30.02% (w
/v) 5% BSA, 0.1% NaN,
17% of 50mM Tris buffer containing 1mM MgCIg
4 volumes were added to prepare an enzyme-labeled antibody solution. In addition, this solution is
It was stored in the dark at 4°C.
■ＥＰＯの測定：
ＥＰＯの測定は、次の様にして行った。抗原コートウェ
ルに被検体液１００μ７！（被検体液は５％ＢＳＡ含有
ＰＢＳ溶？＆、）で適切濃度に希釈した後、１／１０容
量の１０倍緩衝液（０，５％Ｔｗｅｅｎ２０．１０ｍＭ
ＥＤＴＡ、０．２％ＮａＮｙ含有ＰＢＳ溶液）を入れ
、室温で２時間反応（放置）した後、Ｐ Ｂ Ｓ−Ｔｗ
ｅｅｎ溶液（０，０５％Ｔｉｖｅｅｎ２０．０．０２％
ＮａＮｔ含有ＰＢＳ溶液）で各ウェルを３回洗浄した。■Measurement of EPO: Measurement of EPO was performed as follows. Sample body fluid 100μ7 in antigen coated well! (The test body fluid is a PBS solution containing 5% BSA?) After diluting to an appropriate concentration, dilute it to an appropriate concentration with 1/10 volume of 10x buffer (0.5% Tween 20.10mM
PBS solution containing EDTA and 0.2% NaNy was added, and after reacting (standing) at room temperature for 2 hours, PBS-Tw
een solution (0.05% Tiveen20.0.02%
Each well was washed three times with NaNt-containing PBS solution).
次に各ウェルに１００〜２００倍希釈酵素標識抗体溶液
を１００μρずつ入れ、室温で２時間反応（放置）させ
た後、上述と同様にＰＢ Ｓ−Ｔｗｅｅｎ溶液で３回ウ
ェルを洗浄し、遊離の酵素標識抗体を除去する。各ウェ
ルに酵素基質液（ｐ−Ｎｉｔｒｏｐｈｅｎｙｌ ｐｈｏ
ｓｐｈａｔｅ ｄｉｓｏｄｉｕｍを２ｍｇ／ｍ ｅにな
る様に、ＬＭ ｄｉｅｔｈａｎｏｌａ＋ｎ１ｎｅ 、
０．５Ｍ Ｍｇｃｔ！、０．０２％ＮａＮ＊ （ｐＨ９
，８）溶液で調製したもの） １００μβずつを入れ、
室温で３０分間反応させた後、各ウェルに３Ｍ ＮａＯ
Ｈ溶液５０μβずつを加え、酵素反応を停止させた後、
イムノリーダーを用いて、４０５ｎｍにおける各ウェル
の吸光度を測定する。被検液中のＥＰＯ溶液は、既知濃
度のＥＰＯを含有する被検体溶液を用いて同時に同じ操
作を行ってウェルにおける吸光度の強度（検量線）との
比較を行うことにより行った。検量線は添付の第１図に
示す如くであり、５〜６０ｍＵ／ｍ／のＥＰＯ濃度の測
定が可能な検量線が得られた。Next, add 100μρ of a 100- to 200-fold diluted enzyme-labeled antibody solution to each well and let it react (leave) for 2 hours at room temperature.The wells were washed 3 times with PBS-Tween solution in the same manner as above to remove free Remove enzyme-labeled antibodies. Add enzyme substrate solution (p-Nitrophenyl pho) to each well.
LM diethanola+n1ne to make sphate disodium 2mg/me,
0.5M Mgct! , 0.02% NaN* (pH 9
, 8) Prepared as a solution) Add 100μβ each,
After incubating for 30 minutes at room temperature, each well was filled with 3M NaO.
After adding 50μβ of H solution to stop the enzyme reaction,
Measure the absorbance of each well at 405 nm using an immunoreader. The EPO solution in the test solution was determined by performing the same operation at the same time using a test solution containing EPO at a known concentration and comparing it with the absorbance intensity (calibration curve) in the well. The calibration curve is as shown in the attached FIG. 1, and a calibration curve was obtained that allows measurement of EPO concentrations of 5 to 60 mU/m/.
上記ＥＰＯ標準検量曲線を基準とし、各種疾患血清中に
含まれるＥＰＯ量を測定した。表１にその結果を示した
が、従来法のラジオイムノアッセイ法において測定した
結果を比較したところ、本方法が非常に有効であること
が示された。The amount of EPO contained in the serum of various diseases was measured using the above EPO standard calibration curve as a reference. The results are shown in Table 1, and a comparison with the results measured using the conventional radioimmunoassay method showed that the present method is very effective.
表 １ （各種血清中のＥＰＯ量の測定）＊ １１．Ｍ
ｉｚｏｇｕｃｈｉ ｅｔ ａｌ、 Ａｃｔａ Ｈａｅｍ
ａｔｏｌ ５０，１５ （１９Ｂ？）実施例２
ＥＰＯの測定：
実施例１の■に記載した方法に準じて行ったが、被検体
液と酵素標識抗体溶液を同時に各ウェルに入れて反応す
る方法を行った。すなわち、各ウェルに被検体液５０μ
β及び酵素標識抗体溶液５０μβずつ入れ、室温で２時
間反応させた後、ＰＢＳ−Ｔｗｅｅｎ溶液（０，０５％
Ｔｗｅｅｎ２０ＸＯ，０２％ＮａＮ３含有ＰＢＳ溶液）
で、各ウェルを３回洗浄し、未反応のＥＰＯ及び酵素標
識抗体を除去した。次に、各ウェルに酵素基質液１００
μｌずつを入れて室温で３０分間反応させた後に、各ウ
ェルに３Ｍ Ｎａ０Ｉｌ溶液５０μρずつを加え、酵素
反応を停止させた後、イムノリーダーを用いて、４０５
ｎｍにおける各ウェルの吸光度を測定した。被検体液中
のＥ Ｐ Ｏ？Ｍ、度に対する４０５ｎｍにおける吸光
度をプロットした標準曲線を第１図に示した。Table 1 (Measurement of EPO amount in various serums) * 11. M
Izoguchi et al, Acta Haem
atol 50,15 (19B?) Example 2 Measurement of EPO: It was carried out according to the method described in Example 1 (■), but with the exception of a method in which the test body fluid and the enzyme-labeled antibody solution were placed in each well at the same time and reacted. went. That is, each well contains 50μ of the test body fluid.
PBS-Tween solution (0.05%
Tween20XO, PBS solution containing 02% NaN3)
Each well was washed three times to remove unreacted EPO and enzyme-labeled antibodies. Next, 100 ml of enzyme substrate solution was added to each well.
After adding 50 μl of 3M Na0Il solution to each well and reacting for 30 minutes at room temperature, after stopping the enzyme reaction, using an immunoreader,
The absorbance of each well in nm was measured. EPO in the sample body fluid? A standard curve in which absorbance at 405 nm is plotted against M, degrees is shown in FIG.
なお、実施例１の■に記載したと同じ方法に従って被検
体液と酵素標識抗体溶液を別々に反応させた場合を比較
として記載した。両者とも同等の検量曲線が得られ、５
〜６０ｍＵ／／！のＥ Ｐ Ｏ９３度の測定が可能であ
った。In addition, a case where the test body fluid and the enzyme-labeled antibody solution were reacted separately according to the same method as described in Example 1, is described as a comparison. Equivalent calibration curves were obtained for both, and 5
~60mU//! It was possible to measure E P O of 93 degrees.
実施例３
本例はＥＰＯの測定におけるモノクローナル抗体の組合
わせの適否を示したものである。Example 3 This example shows the suitability of a combination of monoclonal antibodies in measuring EPO.
実施例２に記載の方法により、支持体結合抗体、酵素標
識抗体の組合わせの適否を確認した。The suitability of the combination of support-bound antibody and enzyme-labeled antibody was confirmed by the method described in Example 2.
支持体結合抗体（−次抗体）、標識抗体（二次抗体）と
して４種の抗体産生ハイブリドーマの適否を、測定感度
、検量曲線の直線性で比較した。The suitability of four types of antibody-producing hybridomas as support-bound antibodies (secondary antibodies) and labeled antibodies (secondary antibodies) was compared in terms of measurement sensitivity and linearity of calibration curves.
全１２組の組合わせを検討した結果、下記表２に示すと
おり一次抗体ＭＣ−Ｒ−６、門Ｃ−Ｒ−２の組合わせが
最適であることが確認された。As a result of examining all 12 combinations, it was confirmed that the combination of primary antibody MC-R-6 and gate CR-2 was optimal as shown in Table 2 below.
表 ２
◎ ： 検量線、感度ともすくれる
○ ： 検量線、感度のいずれかにやや問題あり△ ：
検量線、感度ともやや問題あり× ： 検量線が引け
ない
λ呪■涜来
以上述べた如く、本発明によれば、既述の構成を採用す
ることによって、従来法における様な放射性同位元素を
用いたり、特殊実験施設を用いることを必要とせず、或
は、煩雑な操作と特殊測定装置（生物発光測定）を要す
ることなく、非常に筒便にＥＰＯの測定が可能となった
。Table 2 ◎: Both the calibration curve and sensitivity are poor ○: There is a slight problem with either the calibration curve or sensitivity △:
There are some problems with both the calibration curve and the sensitivity. It has become possible to measure EPO in a very convenient manner, without using special laboratory facilities or complicated operations and special measuring equipment (bioluminescence measurement).
【図面の簡単な説明】
第１図は、本発明における反応操作をより簡便化した場
合のＥＰＯ測定の標準曲線を簡便化を行わない場合を比
較したグラフである。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph comparing a standard curve for EPO measurement when the reaction operation in the present invention is simplified with a standard curve when the reaction operation is not simplified.
Claims (1)
種のもモノクローナル抗体を選択し、エリスロポエチン
と特異的に反応する第１抗体を固相担体に結合してなる
抗体結合固相担体に、エリスロポエチンを含む被検液を
加えて反応させて該担体に結合させ、次いで、該エリス
ロポエチン結合抗体結合固相担体に前記第１抗体とは異
なる抗原決定部位を有する第２抗体に標識物質を結合さ
せた標識抗体を反応させて上記抗体結合固相担体上に結
合した該標識抗体の量を測定することを特徴とするエリ
スロポエチンの免疫学的測定法。（２）標識物質はアル
カリフォスファターゼである請求項（１）に記載の測定
法。(1) Two different epitopes for erythropoietin
A monoclonal antibody is selected, and a test solution containing erythropoietin is added to an antibody-bound solid phase carrier formed by binding a first antibody that specifically reacts with erythropoietin to a solid phase carrier, and reacted with the carrier. Then, a labeled antibody prepared by binding a labeling substance to a second antibody having an antigen-determining site different from that of the first antibody is reacted with the erythropoietin-bound antibody-bound solid phase carrier to form a labeled antibody on the antibody-bound solid phase carrier. An immunoassay method for erythropoietin, which comprises measuring the amount of bound labeled antibody. (2) The measuring method according to claim (1), wherein the labeling substance is alkaline phosphatase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63079868A JP2657816B2 (en) | 1988-03-31 | 1988-03-31 | Immunoassay for erythropoietin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63079868A JP2657816B2 (en) | 1988-03-31 | 1988-03-31 | Immunoassay for erythropoietin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01250861A true JPH01250861A (en) | 1989-10-05 |
| JP2657816B2 JP2657816B2 (en) | 1997-09-30 |
Family
ID=13702187
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63079868A Expired - Lifetime JP2657816B2 (en) | 1988-03-31 | 1988-03-31 | Immunoassay for erythropoietin |
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| Country | Link |
|---|---|
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5716355A (en) * | 1980-04-25 | 1982-01-27 | Hoffmann La Roche | Immunological method |
| JPS57179750A (en) * | 1981-04-13 | 1982-11-05 | Hoechst Co American | Single culture immunity chemical examining method |
-
1988
- 1988-03-31 JP JP63079868A patent/JP2657816B2/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5716355A (en) * | 1980-04-25 | 1982-01-27 | Hoffmann La Roche | Immunological method |
| JPS57179750A (en) * | 1981-04-13 | 1982-11-05 | Hoechst Co American | Single culture immunity chemical examining method |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2657816B2 (en) | 1997-09-30 |
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